PKC-epsilon is a driving force in phorbol ester (PE)-mediated tumor promotion/ progression and PKC-delta robustly opposes the response in transgenic mouse models. Reinforcing these findings, cell culture models \ have defined PKC-epsilon as growth-stimulatory and oncogenic and PKC-delta as pro-apoptotic arid tumor suppressive. These observations offer a rationale for cancer prevention strategies that inactivate PKC-epsilon or stimulate PKC-delta activity. Recent studies in the PI's laboratory have revealed that disulfide forms of physiological and other thiols have oxidative regulatory effects on PKC isozymes consonant with cancer prevention. For example, cystine inactivates PKC-epsilon and stimulates PKC-delta by S-thiolating the isozymes. In addition to PKC-epsilon, the oncogenic isozymes PKC-beta1 and PKC-gamma are inactivated by S-thiolation. Variable sensitivities of these isozymes to inactivation by a given S-thiolating agent affirmed potential for isozyme-selective PKC targeting, and low S-thiolation stoichiometries provided evidence that each isozyme contains a single Cys switch governing regulation. The goal of this application is to test the hypothesis that PKC isozyme regulation by S-thiolation pivots upon Cys switches that can be exploited as molecular targets for cancer prevention, with focus on the PKC-delta-stimulatory and PKCepsilon-inactivating Cys switches. In Aim 1, S-thiolation-regulated Cys switches will be identified in PKCdelta and PKC-epsilon by site specific mutagenesis. Aim 2 will structurally characterize oxidative modifications transducing S-thiolation-triggered PKC-delta and PKC-epsilon regulation by HPLC and mass spectrometry. Aim 3 will define the influence of Cys switch-mediated regulation on PKC-epsilon-mediated proliferative signaling and PKC-delta- mediated apoptotic signaling. Aim 4 will characterize the in vivo reactivity of Cys switches in PKC isozymes, by determining whether topically applied thiol antioxidants foster S-thiolation-triggered PKC-delta and PKC-epsilon regulation in the DMBA/TPA mouse skin model.